Integrated sample preparation systems and stabilized enzyme mixtures

ABSTRACT

The present invention provides integrated sample preparation systems and stabilized enzyme mixtures. In particular, the present invention provides microfluidic cards configured for processing a sample and generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other suitable nucleic acid analysis methods. The present invention also provides stabilized enzyme mixtures containing an enzyme (e.g., an enzyme used in whole genome amplification), BSA, and a sugar. Such enzyme mixtures may be lyophilized and stored at room temperature without significant loss of enzyme activity for months.

The present application is a continuation of U.S. patent applicationSer. No. 13/102,520 filed May 6, 2011, which claims priority to PCTPatent Application No. PCT/US2011/035597 filed May 6, 2011 and U.S.Provisional Patent application No. 61/331,910 filed May 6, 2010, theentirety of each of which is herein incorporated by reference in itsentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support undercontract number HDTRA-1-07-C-0096. The United States Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to integrated sample preparation andsequencing systems and stabilized enzyme mixtures. In particular, thepresent invention provides microfluidic cards configured for processinga sample and generating DNA libraries that are suitable for use insequencing methods (e.g., next generation sequencing methods) or othersuitable nucleic acid analysis methods, where the output from thesecards can be integrated with a DNA sequencing system providing anautomated and integrated sample to sequence system. The presentinvention also provides stabilized enzyme mixtures containing an enzyme(e.g., an enzyme used in whole genome amplification), BSA, and a sugar.Such enzyme mixtures may be lyophilized and stored at room temperaturewithout significant loss of enzyme activity for months.

BACKGROUND

Sequencing of DNA requires large amounts of extracted DNA for use in thepreparation of a sequencing library. The process of culturing cells,lysing the cells, extracting DNA, fragmenting the DNA, ligating linkers,and purification of the sequencing template is a multi-step process thatcan take several days to be performed by a skilled research technician.The challenge of sequence based detection is that existing whole genomesequencing systems such as the Roche 454 require several days of samplepreparation and several days for sequencing. FIG. 1 shows a flow chartdemonstrating the lengthy process now involved in sample preparation fornext-generation sequencing techniques, such as the Roche 454 method. Asshown in this figure, it can take one day for sample pretreatment; celllysis; nucleic acid extraction; and whole genome amplification (WGA). Itcan then take one to five days for generating DNA libraries, whichinvolves the following steps: DNA fragmentation; DNA end repair; adaptorligation; fragment immobilization; nick repair; single-strand DNAisolation; and emulsion PCR titration. Finally, it can then take one tofour days to prepare the sample for sequencing and the sequencing itselfusing the following steps: bulk emulsion PCR; break emulsion PCR; purifyPCR positive beads; prepare beads for sequencing; and performing thesequencing reaction.

What is needed are methods for preparing DNA sequencing libraries thatare faster and easier to perform and the integration of these methodswith the sequencers.

SUMMARY OF THE INVENTION

The present invention provides integrated sample preparation/nucleicacid sequencing systems and stabilized enzyme mixtures. In particular,the present invention provides microfluidic cards configured forprocessing a sample and generating DNA libraries that are suitable foruse in sequencing methods (e.g., next generation sequencing methods) orother suitable nucleic acid analysis methods. The present invention alsoprovides stabilized enzyme mixtures containing an enzyme (e.g., anenzyme used in whole genome amplification), BSA, and a sugar. Suchenzyme mixtures may be lyophilized and stored at room temperaturewithout significant loss of enzyme activity for months.

In certain embodiments, the present invention provides microfluidiccards comprising: a) a loading port configured for introduction of abiological sample; b) a fragmentation sub-circuit comprising: i) areagent mixture configured for digesting nucleic acid (e.g., amplifiednucleic acid) to generate fragmented nucleic acid, and/or ii) afragmentation component configured for mechanically fragmenting nucleicacid to generate fragmented nucleic acid; and c) a linker ligationsub-circuit operably linked to said fragmentation sub-circuit, whereinsaid linker ligation sub-circuit comprises: i) nucleic acid linkersconfigured for use in sequencing methods or other methods, and ii) aligation enzyme mixture configured for ligating said nucleic acidlinkers to said fragmented nucleic acid to generate a nucleic acidsequencing library.

In certain embodiments, the present invention provides microfluidiccards comprising: a) a loading port configured for introduction of abiological sample; b) a cell lysis subcircuit; c) as nucleic acidextraction subcircuit; d) a nucleic acid amplification subcircuitwherein said amplification subcircuit comprises i) a target sequencespecific amplification such as PCR or ii) a total nucleic acidamplification subcircuit such as whole genome amplification by multipledisplacement amplification; e) a fragmentation sub-circuit comprising:i) a reagent mixture configured for digesting nucleic acid (e.g.,amplified nucleic acid) to generate fragmented nucleic acid, and/or ii)a fragmentation component configured for physically fragmenting nucleicacid to generate fragmented nucleic acid; f) end polishing of fragmentednucleic acid; g) a linker ligation sub-circuit operably linked to saidfragmentation sub-circuit, wherein said linker ligation sub-circuitcomprises: i) nucleic acid linkers configured for use in sequencingmethods or other methods, and ii) a ligation enzyme mixture configuredfor ligating said nucleic acid linkers to said fragmented nucleic acidto generate a nucleic acid sequencing library; h) methods for theremoval of non-ligated or partially ligated nucleic acids; i) This canbe performed enzymatically using for example exonucleases and or ii) bybind and elute extraction or iii) by affinity isolations using affinitytags as biotin labeled ligation products; i) final library purificationby enzymatic or bind elute or affinity or size exclusion etc; j)integration with a sequencing system.

In particular embodiments, the microfluidics cards further comprise atleast one additional sub-circuit selected from the following group: 1) alysis sub-circuit (e.g., operably linked to said loading port)comprising: i) a mixing chamber and, ii) lysis buffer (e.g., in a sealedpackage); 2) a nucleic acid extraction sub-circuit (e.g., operablylinked to both said lysis sub-circuit and a waste chamber), wherein saidnucleic acid extraction sub-circuit comprises: i) a nucleic acidextraction component configured to bind nucleic acids present in a lysedsample, ii) a wash buffer (e.g., in a sealed package), iii) an elutionbuffer (e.g., in a sealed package), and iv) a pump component (e.g.,configured for pumping the elution buffer over the nucleic acidextraction component to generate a mixture of extracted nucleic acids);3) an amplification sub-circuit (e.g., operably linked to the nucleicacid extraction sub-circuit) comprising a stabilized enzyme mixture,wherein said stabilized enzyme mixture comprises at least oneamplification-related enzyme useful for performing amplification on theextracted nucleic acid to generate amplified nucleic acid; and 4) awaste chamber;

In certain embodiments, the present invention provides microfluidiccards comprising: a) a loading port configured for introduction of abiological sample; b) an amplification sub-circuit (e.g., operablylinked to the nucleic acid extraction sub-circuit) comprising astabilized enzyme mixture, wherein said stabilized enzyme mixturecomprises at least one amplification-related enzyme useful forperforming whole genome amplification on nucleic acids to generateamplified nucleic acid.

In some embodiments, the present invention provides microfluidic cardscomprising: a) a loading port configured for introduction of abiological sample; b) a waste chamber; c) a cell lysis sub-circuitoperably linked to the loading port, wherein the cell lysis sub-circuitcomprises: i) a mixing chamber and, ii) a first sealed packagecontaining lysis buffer; wherein the lysis sub-circuit is configured tolyse the biological sample in the mixing chamber with the lysis bufferto generate a lysed sample; d) a nucleic acid extraction sub-circuitoperably linked to both the cell lysis sub-circuit and the wastechamber, wherein the nucleic acid extraction sub-circuit comprises: i) anucleic acid extraction component configured to bind nucleic acidspresent in the lysed sample, ii) a second sealed package containing washbuffer, iii) a third sealed package containing elution buffer, and iv) apump component configured for pumping the elution buffer over thenucleic acid extraction component to generate a mixture of extractednucleic acids; e) an amplification sub-circuit operably linked to thenucleic acid extraction sub-circuit, wherein the amplificationsub-circuit comprises a stabilized enzyme mixture, wherein thestabilized enzyme mixture comprises at least one amplification-relatedenzyme useful for performing amplification on the extracted nucleic acidto generate amplified nucleic acid; f) a fragmentation sub-circuitoperably linked to the amplification sub-circuit, wherein thefragmentation sub-circuit comprises: i) a reagent mixture configured fordigesting the amplified nucleic acid to generate fragmented nucleicacid, and/or ii) a fragmentation component configured for mechanicallyfragmenting the amplified nucleic acid to generate fragmented nucleicacid; and g) a linker ligation sub-circuit operably linked to thefragmentation sub-circuit, wherein the linker ligation sub-circuitcomprises: i) nucleic acid linkers (adaptors) configured for use insequencing methods, and ii) a ligation enzyme mixture configured forligating the nucleic acid linkers to the fragmented nucleic acid togenerate a nucleic acid sequencing library.

In certain embodiments, the at least one amplification-related enzymecomprises an enzyme useful for performing whole genome amplification(WGA) (e.g., multiple displacement amplification), or useful forperforming PCR, or useful for performing transcription mediatedamplification (TMA). In some embodiments, the at least oneamplification-related enzyme is selected from the group consisting of:Phi-29 polymerase, E. coli DNA polymerase I, inorganic pyrophosphatase,or any combination thereof. In certain embodiments, the amplificationrelated enzymes are combined with linkers (adapters) that are used innext-generation sequencing, such as the next-generation sequencingmethods described herein. In particular embodiments, the primer used forany amplification step (e.g., PCR) comprise linkers that are useful innext-generation sequencing methods (e.g., for tethering the amplicons toa solid support).

In particular embodiments, the stabilized enzyme mixture furthercomprises: i) BSA, ii) a sugar, and iii) at least one additionalcomponent selected from the group consisting of: an inorganic salt, adivalent metal cation, a buffering agent, an emulsifier, and a reducingagent. In other embodiments, the stabilized enzyme mixture furthercomprises: i) BSA, ii) a sugar, iii) an inorganic salt, iv) a divalentmetal cation, v) a buffering agent, vi) an emulsifier, and vii) areducing agent.

In some embodiments, the microfluidic cards further comprise: h) apurification sub-circuit operably linked to the linker ligationcomponent, wherein the purification sub-circuit comprises a nucleic acidpurification component, wherein the nucleic acid purification componentcomprises anchored nucleic acid sequences configured to hybridize to thelinkers on the nucleic acid sequencing library. In other embodiments,the microfluidic cards further comprise an outlet port configured toallow a user to withdraw at least a portion of the nucleic acidsequencing library. In certain embodiments, the fragmentation subcircuitfurther comprises: at least one type of enzyme configured for polishingthe ends of the fragmented nucleic acid. In particular embodiments, thelinker ligation sub-circuit further comprises: at least one type ofenzyme configured for polishing the ends of the nucleic acid sequencinglibrary. In other embodiments, the stabilized enzyme mixture is presentin a dried format. In some embodiments, the reagent mixture is presentin a dried format.

In further embodiments, the pump component comprises a bellows. In otherembodiments, the nucleic acid extraction components comprises amembrane. In additional embodiments, the nucleic acid extractioncomponents comprises a filter. In particular embodiments, themicrofluidics cards comprise a plurality of valves. In additionalembodiments, the card is configured to operably link to, and be operatedby, a processing instrument. In other embodiments, the microfluidicscards further comprise a plurality of air ports configured to operablylink with a pneumatic interface on the processing instrument. In someembodiments, the nucleic acid linkers are configured for use in asequencing method selected from the group consisting of: ABI SOLID,ILLUMINA SOLEXA, ROCHE 454, ION TORRENT, Lifetechnologies STARLITE, andPACIFIC BIOSCIENCES SMRT sequencing. In additional embodiments, themicrofluidics cards further comprise a sensor configured to determine ifthe mixture of extracted nucleic acids requires amplification or not.

The sample preparation systems of the present invention may beintegrated with any type of sequencing system (e.g., as a re-usable cardor disposable card). In certain embodiments, the sample preparationsystems of the present invention are integrated with ILLIMINA SOLEXAsequencers including HiSeq 2000, HiSeq 1000, HiScanSQ, Genome AnalyzerIIx, MiSeq, where the sample preparation systems may be configured towork with the related Illumina software, such as the PIPELINE and/orCASAVA software packages. In other embodiments, the sample preparationsystems of the present invention are integrated with the ROCHE 454sequencers, including the Genome Sequencer FLX System and GS JuniorSystem, where the sample preparation systems may be configured to workwith the related GS Run Browser Software, GS De Novo Assembler Software,GS Reference Mapper Software, GS Amplicon Variant Analyzer Software, andthe GS FLX Titanium Cluster. In some embodiments, the sample preparationsystems of the present invention are integrated with the ION TORRENTsequencers, including the Ion Personal Genome Machine (PGM) sequencer,where the sample preparation systems may be configured to work with therelated DNASTAR® SeqMan® NGen® Software, Partek® Genomics Suite™Software, NextGENe® software for Ion PGM platform by SoftGenetics, orAvadis NGS Software. In additional embodiments, the sample preparationsystems of the present invention are integrated with the PACIFICBIOSCIENCES sequencers, including the PacBio RS sequencer, where thesample preparation systems may be configured to work with the related RSremote software, RS touch software, Primary Analysis software, SMRTPortal software, and SMRT View software.

In some embodiments, the present invention provides systems comprising:a microfluidic card as described herein; and b) a processing instrumentconfigured to receive and operate the microfluidics card. In certainembodiments, the processing instrument comprises at least one componentselected from: a pressure reservoir, a vacuum reservoir, at least onepump, a plurality of valves, at least one heater, a pneumatic interface,and an input-output computer connection. In additional embodiments, theprocessing instrument comprises: a pressure reservoir, a vacuumreservoir, at least one pump, a plurality of valves, at least oneheater, a pneumatic interface, and an input-output computer connection.

In some embodiments, the present invention provides stabilized enzymemixtures comprising, or consisting essentially of, or consisting of: a)at least one type of enzyme; b) bovine serum albumin (BSA); c) a sugar;and d) a buffering agent; and optionally water. In certain embodiments,the stabilized enzyme mixture further comprises, or consists essentiallyof, or consists of: at least one reagent selected from the groupconsisting of: an inorganic salt; a divalent metal cation; anemulsifier; and a reducing agent.

In particular embodiments, the present invention provides stabilizedenzyme mixtures comprising, or consisting essentially of, or consistingof: a) at least one type of enzyme; b) bovine serum albumin (BSA); c) asugar; d) an inorganic salt; e) a divalent metal cation; f) a bufferingagent; g) an emulsifier; and h) a reducing agent.

In certain embodiments, the mixtures further comprise polyethyleneglycol (PEG) (e.g., PEG-8000 or other weights). In further embodiments,the stabilized enzyme mixture is in an aqueous form or in a lyophilizedform. In other embodiments, the stabilized enzyme mixture allows theenzyme to retain at least 70% of its activity (e.g., 70% . . . 75% . . .80% . . . 85% . . . 90% . . . 95% . . . 100%) upon hydration at 20-25degrees Celsius after storage for two months at 20-40 degrees Celsius.

In further embodiments, the BSA is present at a concentration of0.05%-3.0% in the stabilized enzyme mixture (e.g., 0.05% . . . 0.10% . .. 0.5% . . . 1.0% . . . 2.0% . . . 3.0%). In further embodiments, thesugar is present at a concentration of 5-35% of the stabilized enzymemixture (e.g., 5% . . . 15% . . . 25% . . . 35%). In other embodiments,the sugar is a non-reducing sugar. In further embodiments, the sugar isa disaccharide. In additional embodiments, the sugar is trehalose.

In some embodiments, the at least one type of enzyme comprises amesophilic enzyme, a thermolabilze enzyme, or a thermophilic enzyme. Inadditional embodiments, the at least one type of enzyme comprises apolymerase. In particular embodiments, the polymerase is Phi-29polymerase. In further embodiments, the polymerase is E. coli PolymeraseI. In additional embodiments, the at least one type of enzyme comprisesan inorganic pyrophosphatase. In certain embodiments, the inorganicpyrophosphatase is Saccharomyces cerevisiae inorganic pyrophosphatase.In additional embodiments, the at least one type of enzyme comprises:Phi-29 polymerase, E. coli. DNa Polymerase I, and S. cerevisiaeinorganic pyrophosphatase.

In certain embodiments, the inorganic salt is present at a concentrationof 1 mM-25 mM in the stabilized enzyme mixture (e.g., 1 mM . . . 10 mM .. . 17 mM . . . 25 mM). In further embodiments, the inorganic salt is(NH₄)₂SO₄. In certain embodiments, the divalent metal cation is presentat a concentration of 1 mM-30 mM in the stabilized enzyme mixture (e.g.,1 mM . . . 10 mM . . . 20 mM . . . or 30 mM). In particular embodiments,the divalent metal cation is MgCl₂. In further embodiments, thebuffering agent is present at a concentration of 10 mM-100 mM (e.g., 10mM . . . 35 mM . . . 75 mM . . . 100 mM). In some embodiments, thebuffering agent is Tris.

In particular embodiments, the emulsifier is present at a concentrationof 0.01%-0.15% in the stabilized enzyme mixture (e.g., 0.01% . . . 0.1%. . . 0.15%). In some embodiments, the emulsifier is Tween 40, Tween 20,or Tween 80. In additional embodiments, the reducing agent is present ata concentration of 1 mM-10 mM (e.g., 1 mM . . . 5 mM . . . 10 mM). Incertain embodiments, the reducing agent is dithiothreitol (DTT).

In some embodiments, the present invention provides compositions forstabilizing an enzyme comprising, consisting essentially of, orconsisting of: a) bovine serum albumin (BSA); b) a sugar; c) aninorganic salt; and optionally 1) a divalent metal cation; 2) abuffering agent; 3) an emulsifier; 4) a reducing agent; and 5) water.

In further embodiments, the present invention provides compositionscomprising, or consisting essentially of, or consisting of: E. colipolymerase I and bovine serum albumin (BSA), wherein the composition isa lyophilized composition. In particular embodiments, the lyophilizedcomposition allows the E. coli polymerase Ito retain at least 70% of itsactivity (e.g., 70% . . . 90% . . . 100%) upon hydration at 20-25degrees Celsius after storage for at least one, two, or three months at20-40 degrees Celsius.

In certain embodiments, the present invention provides compositionscomprising, or consisting essentially of, or consisting of: inorganicphosphatase and bovine serum albumin (BSA), wherein the composition is alyophilized composition. In some embodiments, the lyophilizedcomposition allows the inorganic phosphatase to retain at least 70%(e.g., 70% . . . 90% . . . 100%) of its activity upon hydration at 20-25degrees Celsius after storage for two months at 20-40 degrees Celsius.

In further embodiments, the present invention provides methods ofstoring a lyophilized composition comprising: a) providing a lyophilizedcomposition comprising, or consisting essentially of, or consisting of:i) at least one type of enzyme; ii) bovine serum albumin (BSA); iii) asugar; iv) an inorganic salt; v) a divalent metal cation; vi) abuffering agent; vii) an emulsifier; and viii) a reducing agent; and b)storing the lyophilized composition at a storage temperature of 15-45degrees Celsius for at least 15 days such that the at least one enzymeretains at least 70% of its activity upon hydration at 20-25 degreesCelsius. In further embodiments, the at least 15 days is at least 30days (e.g., at least 30 days . . . 60 days . . . 90 days . . . 120 days. . . or longer). In certain embodiments, the storage temperature is20-25 degrees Celsius. In some embodiments, the at least one enzyme isselected from E. coli polymerase I, Phi-29 polymerase, and inorganicpyrophosphatase.

In particular embodiments, the present invention provides methods ofgenerating a stabilized enzyme composition comprising: a) providing: i)an aqueous composition comprising, or consisting essentially of, orconsisting of: A) at least one type of enzyme; B) bovine serum albumin(BSA); C) a sugar; D) an inorganic salt; E) a divalent metal cation; F)a buffering agent; G) an emulsifier; and H) a reducing agent; and ii) adializing membrane; and b) dialyzing the aqueous composition with thedialzying membrane into a solution comprising or consisting essentiallyof: i) the sugar; ii) the inorganic salt; iii) the divalent metalcation; iv) the buffering agent; v) the emulsifier; and vii) thereducing agent; c) freezing the aqueous composition to generate a frozencomposition; and d) subjecting the frozen composition to high vacuum toremove water via sublimation such that a lyophilized composition isgenerated.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a flow chart demonstrating the lengthy process now involvedin sample preparation for next-generation sequencing techniques, such asthe Roche 454 method.

FIG. 2 shows an exemplary injected molded microfluidic card and thevarious layers that may make up a card. FIG. 2 also describes the firstthree sub-circuits of cell lysis, DNA extraction, and whole genomeamplification.

FIG. 3 shows an exemplary microfluidic card with various sub-circuitslabeled, including a sample inlet port, a DNA extraction circuit, and anamplification circuit which includes lyophilized enzymes (e.g., such asthose described below in Example 1).

FIG. 4 shows step 1 of a sample preparation process utilizing amicrofluidic card. This figure specifically shows a sample inlet portand a mixing chamber.

FIG. 5 shows step 2 which is a lysis step. Lysis buffer from a blisterpack flows into a mixing chamber with cells from the sample such thatthe cells are lysed.

FIG. 6 shows step 3 where the lysed mixture is passed over a capturefilter and then to a waste chamber. A first wash buffer from a blisterpack is then passed over the filter before going into the waste chamber.

FIG. 7 shows step 4 where a second wash is passed through the capturefilter and then on to the waste chamber. The filter is then air dried toremove the wash buffer.

FIG. 8 shows step 5 where an elution buffer from a blister pack removesthe purified nucleic acid from the filter using an elution bellows. Theelution bellows pumps the elution buffer slowly over the filter.

FIG. 9 describes the three reference samples (S. aureus; B. cereus; andK. pneumoniae) that were used during development of embodiments of thepresent invention in order to evaluate the lysis and extraction steps.

FIG. 10 shows the results of final validation of the lysis andextraction sub-circuits using S. aureus; B. cereus; and K. pneumoniae.The results shown are for inputs of 10,000 CFUs of B. cereus and K.pneumoniae, and freshly grown cultures of S. aureus.

FIG. 11 shows production of contamination-free whole genomeamplification reagents.

FIG. 12 shows step 6 where the purified nucleic acid sample is passedinto the amplification sub-circuit with the reagents (includingstabilized enzyme mixture lyophilized beads) inside the card.

FIG. 13 shows an exemplary microfluidic card with the reagents(including stabilized enzyme mixture lyophilized beads) inside the card,as well as the configuration of the buffer packs.

FIG. 14 describes that prior art WGA enzyme mixtures were not stableduring storage, with stability for only 5 days at room temperature. FIG.14 also describes the advantages of lyophilized mixtures ofamplification enzymes, which are provided in Example I below.

FIG. 15 shows that many excipient formulations were tested (inExample 1) for compatibility with Phi-29, Polymerase I, and inorganicpyrophosphatase.

FIG. 16 shows results from Example 1 indicating that BSA is an importantcomponent for stabilizing amplification enzymes.

FIG. 17 shows that a number of BSA levels (0.13%-1% final concentration)as well as the addition of 0.5% (final concentration) of PEG-8000 weretested in the enzyme stabilization formulations as described in Example1.

FIG. 18 shows that the lyophilized enzymes in Ibis Formula 33 whenstored at room temperature for 4 months performed equal to fresh enzyme.

FIG. 19 shows that the lyophilized enzymes in Ibis Formula 33 performedequal to fresh enzyme after two months at 40 degrees Celsius.

FIG. 20 shows an exemplary microfluidic card including a moldedcartridge (with a waste pad and liquid reagents), a top lid, a laminatebottom, and a pneumatic gasket.

FIG. 21 shows an image of an exemplary buffer blister pack. FIG. 21describes how molded sharps on the microfluidic card can be used topuncture the blister packs and release their stored reagents.

FIG. 22 shows a schematic of an exemplary processing instrument used tointerface with and operate the microfluidic cards.

FIG. 23 shows an exemplary system overview of a consumable microfluidiccard working with the processing instrument.

FIG. 24 shows exemplary results of fragmenting an amplified sample witheither physical or enzymatic means.

DEFINITIONS

As used herein, the phrase “microfluidic card” refers to a device,cartridge or “card” with selected internal channels, voids or othermicrostructures having at least one dimension on the order of 0.1 to 500microns. Microfluidic devices may be fabricated from various materialsusing techniques such as laser stenciling, embossing, stamping,injection molding, masking, etching, and three-dimensional softlithography. Laminated microfluidic devices are further fabricated withadhesive interlayers or by thermal adhesiveless bonding techniques, suchas by pressure treatment of oriented polypropylene. Themicroarchitecture of laminated and molded microfluidic devices candiffer. In certain embodiments, the microfluidic cards of the presentinvention are designed to interact or “dock” with a host instrument thatprovides a control interface and optional temperature and magneticinterfaces. The card, however, generally contains all biologicalreagents needed to perform the assay and requires only application of asample or samples. These cards are generally disposable, single-use, andare generally manufactured with sanitary features to minimize the risksof exposure to biohazardous material during use and upon disposal.

The term “whole genome amplification” or “WGA” as used herein generallyrefers to a method for amplification of a limited DNA sample in anon-specific manner (unless targeted WGA is employed), in order togenerate a new sample that is indistinguishable from the original butwith a higher DNA concentration. The ideal whole genome amplificationtechnique would amplify a sample up to a microgram level whilemaintaining the original sequence representation. The DNA of the samplemay include an entire genome or a portion thereof. Degenerateoligonucleotide-primed PCR (DOP), primer extension PCR technique (PEP)and multiple displacement amplification (MDA), are examples of wholegenome amplification methods.

The term “multiple displacement amplification” as used herein, refers toa non-PCR-based isothermal method based on the annealing of randomhexamers (or non-random primers in targeted methods) to denatured DNA,followed by strand-displacement synthesis at constant temperature. Ithas been applied to small genomic DNA samples, leading to the synthesisof high molecular weight DNA with limited sequence representation bias.As DNA is synthesized by strand displacement, a gradually increasingnumber of priming events occur, forming a network of hyper-branched DNAstructures. The reaction can be catalyzed by, for example, the Phi29 DNApolymerase or by the large fragment of the Bst DNA polymerase.

DETAILED DESCRIPTION

The present invention provides integrated sample preparation systems andstabilized enzyme mixtures. In particular, the present inventionprovides microfluidic cards configured for processing a sample andgenerating DNA libraries that are suitable for use in sequencing methods(e.g., next generation sequencing methods) or other suitable nucleicacid analysis methods. The present invention also provides stabilizedenzyme mixtures containing an enzyme (e.g., an enzyme used in wholegenome amplification), BSA, and a sugar. Such enzyme mixtures may belyophilized and stored at room temperature without significant loss ofenzyme activity for months.

I. Microfluidic Cards and Instruments

The present invention provides for the integration of several molecularbiology processes/steps into a single integrated system using amicrofluidic card. The integrated system can then, for example, be usedto take samples (e.g., clinical, biological, environmental) and lyse thecells, extract the nucleic acids, amplify the extracted nucleic acids,(e.g., whole genome amplify), fragment the amplified nucleic acid,polish the DNA fragment ends, ligate linkers and purify the processednucleic acid (e.g., a DNA sequencing library suitable for sequencing).The integrated process dramatically reduces the processing time, laborand improves the consistency of the process through the use ofautomation and quality controlled reagents that are integrated into thesingle use system.

The entire process can be integrated into single use microfluidic cardthat contains all the reagents needed to carry out the process. In someembodiments, the process wastes are also contained within the cards. Thereagents can be stabilized so that the cards can be stored at roomtemperature for long periods of time. The cards generally contain a portwhere the resulting processed can be removed and used with a variety ofDNA sequencing technologies such as Sanger sequencing, ABI SOLID,Illumina Solexa, Roche 454, Ion Torrent, ABI Starlite, PacBio SMRT, andother nucleic acid analysis techniques.

In certain embodiments, the microfluidic cards manufactured by MicronicsInc. and described in their patent publications are employed as part ofthe present invention. Micronics' PanNAT™ molecular diagnostic platformis described as a convenient, battery and/or main powered instrumentcapable of processing distinct cartridges, each designed to perform asingle and/or multiplexed nucleic acid amplification assay. Each assayis fully integrated into the disposable cartridge that includes allnecessary reagents. Only a small volume of biological sample is requiredfor assay performance. A description of certain Micronics cards areprovided in U.S. Patent Publication 20090325276, which is hereinincorporated by reference in its entirety as if fully set forth herein.In certain embodiments, the microfluidic cards employed with the presentinvention are as described in the following paragraphs.

Microfluidic cards are formed from multiple subcircuits corresponding toindependent assay modules, but integrated together in a single device ortwo or more interconnected devices. Each subcircuit in turn ispreferably made up of microfluidic elements or components. Elements ofthese subcircuits may include microfluidic channels, tees, chambers,valves, filters, solid phase capture elements, isolation filters,pneumatic manifolds, blister packs (e.g., with reagent pouches), wastesequestration chambers, sanitary vents, bellows chambers, bellows pumps,optical windows, test pads, and microchannel-deposits of dehydratedreagents, optionally including buffers, solubilizers, and passivatingagents. The subcircuits are generally fabricated of plastic, and may bemade by lamination, by molding, and by lithography, or by a combinationof these technologies.

The card devices are typically single-entry, meaning that after a sampleor samples are introduced, the device is sealed so that any potentialbiohazard is permanently entombed in the card for disposal. However, incertain embodiments, the cards have an outlet port for removal ofresulting DNA libraries. The cards are typically self-contained, in thatany reagents needed for the assay are supplied with the device by themanufacturer. It is understood that microfluidic devices optionally mayinclude RFID, microchips, bar codes, and labeling as an aid inprocessing analytical data and that the host instrument for card dockingis optionally a smart instrument and can communicate patient data andtest results to a network.

Microfluidic channels also termed “microchannels,” are fluid channelshaving variable length, but one dimension in cross-section is less than500 um. Microfluidic fluid flow behavior in a microfluidic channel ishighly non-ideal and laminar and may be more dependent on wall wettingproperties, roughness, liquid viscosity, adhesion, and cohesion than onpressure drop from end to end or cross-sectional area. The microfluidicflow regime is often associated with the presence of “virtual liquidwalls” in the channel. Microfluidic channels are fluidly connected by“tees” to each other or to other process elements. Valves are formed inmicrofluidic channels, and may be check valves, pneumatic check valves,pinch valves, surface tension valves, and the like, as conventionallyused.

The card devices generally contain an overlying pneumatic manifold thatserves for control and fluid manipulation, although electronicallyactivated valves could also be used. Air ports are connected to thepneumatic manifold, and generally activate bellows pumps. Where thevalves are pneumatically actuated, air ports are also implicated. Airports are sometimes provided with hydrophobic isolation filters (e.g.,any liquid-impermeable, gas-permeable filter membrane) where leakage offluid from within the device is undesirable and unsafe. Vents are notgenerally directly connected to the pneumatic manifold, but serve toequalize pressures within it.

Reaction chambers are provided on the microfluidic cards and can be anysuitable shape, such as rectangular chambers, circular chambers, taperedchambers, serpentine channels, and various geometries for performing areaction. These chambers may have windows for examination of thecontents, as in detection chambers. Waste sequestration receptacles aregenerally provided on the microfluidic cards. Waste receptacles areoptionally vented with sanitary hydrophobic membranes.

FIG. 2 shows an exemplary injected molded microfluidic card and thevarious layers that may make up a card. FIG. 2 also describes the firstthree sub-circuits of cell lysis, DNA extraction, and whole genomeamplification.

FIG. 3 shows an exemplary microfluidic card with various sub-circuitslabeled, including a sample inlet port, a DNA extraction circuit, and anamplification circuit which includes lypholized enzymes (e.g., such asthose described below in Example 1).

FIG. 4 shows step 1 of a sample preparation process utilizing amicrofluidic card. This figure specifically shows a sample inlet portand a mixing chamber.

FIG. 5 shows step 2 which is a lysis step. Lysis buffer from a blisterpack flows into a mixing chamber with cells from the sample such thatthe cells are lysed.

FIG. 6 shows step 3 where the lysed mixture is passed over a capturefilter and then to a waste chamber. A first wash buffer from a blisterpack is then passed over the filter before going to the waste chamber.

FIG. 7 shows step 4 where a second wash is passed through the capturefilter and then on to the waste chamber. The filter is then air dried toremove the wash buffer.

FIG. 8 shows step 5 where an elution buffer from a blister pack removesthe purified nucleic acid from the filter using an elution bellows. Theelution bellows pumps the elution buffer slowly over the filter.

FIG. 9 describes the three reference samples (S. aureus; B. cereus; andK. pneumoniae) that were used during development of embodiments of thepresent invention in order to evaluate the lysis and extraction steps.

FIG. 10 shows the results of final validation of the lysis andextraction sub-circuits using S. aureus; B. cereus; and K. pneumoniae.The results shown are for inputs of 10,000 CFUs of B. cereus and K.pneumoniae, and freshly grown cultures of S. aureus.

FIG. 11 production of contamination-free whole genome amplificationreagents. In certain embodiments, reagents used for WGA are passedthrough a DNA absorbing unit and a 0.2 uM sterile filter. The resultsare purified reagents, where the negative controls do not produce DNAeven after 12 hours.

FIG. 12 shows step 6 where the purified nucleic acid sample is passedinto the amplification sub-circuit. In this circuit, whole genomeamplification can be performed using a zone where the purified samplemixes with the amplification buffer (which can be the same as theelution buffer) and heated to 95 degrees Celsius and then moved to areaction zone where it mixes with the amplification enzymes at 30degrees Celsius. Preferably, the enzymes are lyophilized amplificationenzymes that are part of stabilized enzyme mixtures as described inExample 1 below.

FIG. 13 shows an exemplary microfluidic card with the reagents(including stabilized enzyme mixture lyophilized beads) dried inside thecard, as well as the configuration of the buffer packs.

FIG. 14 describes that prior art WGA enzyme mixtures were not stableduring storage, with stability for only 5 days at room temperature. FIG.14 also describes the advantages of lyophilized mixtures ofamplification enzymes, which are provided in Example 1 below.

FIG. 20 shows an exemplary microfluidic card including a moldedcartridge (with a waste pad and liquid reagents), a top lid, a laminatebottom, and a pneumatic gasket.

FIG. 21 shows an image of an exemplary buffer blister pack. FIG. 21describes how molded sharps on the microfluidic card can be used topuncture the blister packs and release their stored reagents.

FIG. 22 shows a schematic of an exemplary processing instrument used tointerface with and operate the microfluidic cards. As described in thisfigure, the processing instrument includes a processor to run theinstrument; an input-output that may include a USB or Ethernetconnection; an LCD screen to provide status to a user; and a touchscreen for user control. The processing instrument may also include apneumatic system containing air pumps, regulators, manifolds,pressure/vacuum reservoirs; and valves. The processing instrument mayalso include a card/cartridge interface with clamps, a pneumaticmanifold, and heaters.

FIG. 23 shows an exemplary system overview of the consumablemicrofluidic card working with the processing instrument.

FIG. 24 shows exemplary results of fragmenting a WGA amplified samplewith either physical or enzymatic means. Such fragmentation may beperformed in step 7 in the microfluidic card in a fragmentationsub-circuit. The amplified sample may be moved to the fragmentationsub-circuit and then be fragmented using mechanical shearing (e.g., bypassing through an orifice in the fragmentation sub-circuit) or may besubjected to restriction enzymes. Fragmentation methods that may beemployed include, but are not limited to, cleavage with restrictionenzymes or cleavage primers, for example. Methods of using restrictionenzymes and cleavage primers are well known to those with ordinary skillin the art. The ends of the fragments may then be polished with enzymes,such as Klenow fragment.

In certain embodiments, the next sub-circuit in the microfluidic cardsis a linker ligation sub-circuit. In this sub-circuit, linkers areligated to the ends of the fragmented nucleic acid. Preferably thelinkers are those used in sequencing methods, such as those used in ABISOLID, ILLUMINA SOLEXA, ROCHE 454, ION TORRENT, ABI STARLITE, andPACIFIC BIOSCIENCES SMRT sequencing. A description of these sequencingtechnologies and associated linkers is provided further below.

II. Whole Genome Amplification Methods

In certain embodiments, the microfluidic cards have reagents necessary,sufficient, or useful to perform whole genome amplification (WGA) in theamplification sub-circuit. It is noted that the present invention is notlimited to WGA as the amplification technology, as any other type ofsuitable amplification technology (and corresponding reagents) maybeemployed, such as PCR or TMA, both of which are well known in the art.

A. Non-Target WGA

In many fields of research such as genetic diagnosis, cancer research orforensic medicine, the scarcity of genomic DNA can be a severelylimiting factor on the type and quantity of genetic tests that can beperformed on a sample. One approach designed to overcome this problem iswhole genome amplification (WGA). The objective is to amplify a limitedDNA sample in a non-specific manner in order to generate a new samplethat is indistinguishable from the original but with a higher DNAconcentration. The aim of a typical whole genome amplification techniquewould be to amplify a sample up to a microgram level while respectingthe original sequence representation.

The first whole genome amplification methods were described in 1992, andwere based on the principles of the polymerase chain reaction. Zhang andcoworkers (Zhang, L., et al. Proc. Natl. Acad. Sci. USA, 1992, 89:5847-5851, herein incorporated by reference) developed the primerextension PCR technique (PEP) and Telenius and collaborators (Teleniuset al., Genomics. 1992, 13(3):718-25, herein incorporated by reference)designed the degenerate oligonucleotide-primed PCR method (DOP-PCR)Zhang et al., 1992).

DOP-PCR is a method which uses Taq polymerase and semi-degenerateoligonucleotides (such as CGACTCGAGNNNNATGTGG (SEQ ID NO: 1), forexample, where N=A, T, C or G) that bind at a low annealing temperatureat approximately one million sites within the human genome. The firstcycles are followed by a large number of cycles with a higher annealingtemperature, allowing for the amplification of the fragments that weretagged in the first step.

Multiple displacement amplification (MDA, also known as stranddisplacement amplification; SDA) is a non-PCR-based isothermal methodbased on the annealing of random hexamers to denatured DNA, followed bystrand-displacement synthesis at constant temperature (Blanco et al.,1989, J. Biol. Chem. 264:8935-40, herein incorporated by reference). Ithas been applied to small genomic DNA samples, leading to the synthesisof high molecular weight DNA with limited sequence representation bias(Lizardi et al., Nature Genetics 1998, 19, 225-232; Dean et al., Proc.Natl. Acad. Sci. U.S.A. 2002, 99, 5261-5266; both of which are hereinincorporated by reference). As DNA is synthesized by stranddisplacement, a gradually increasing number of priming events occur,forming a network of hyper-branched DNA structures. The reaction can becatalyzed by the Phi29 DNA polymerase or by the large fragment of theBst DNA polymerase. The Phi29 DNA polymerase possesses a proofreadingactivity resulting in error rates 100 times lower than the Taqpolymerase.

In some embodiments, the reaction mixtures employed for the multipledisplacement amplification include a plurality of polymerase enzymes. Insome embodiments, the catalytic activities include 5′-3′ DNA polymeraseactivity, 3′-5′ exonuclease proofreading activity, and DNA repairactivities such as, for example, 5′-3′ excision repair activity.Examples of various polymerase enzymes include, but are not limited to,the following: Phi29, Klenow fragment, T4 polymerase, T7 polymerase,BstE polymerase, E. coli Pol I, Vent, Deep Vent, Vent exo-, Deep Ventexo-, KOD HiFi, Pfu ultra, Pfu turbo, Pfu native, Pfu exo-, Pfu exo-Cx,Pfu cloned, PROOFSTART (Qiagen), rTth, Tgo and Tfu Qbio. Thesepolymerases are known and most are commercially available.

In other embodiments, other non-polymerase enzymes or accessory proteinsare included in the MDA reaction mixtures such as, for example,helicase, gyrase, T4G32 and SSBP for example. In some embodiments, thereaction mixture for MDA includes pyrophosphatase which serves toconvert pyrophosphate to phosphate. Pyrophosphate accumulates in thereaction mixture as a result of the amplification reaction (oneequivalent of pyrophosphate is generated from each incorporateddeoxynucleotide triphosphate added and is known to inhibit theamplification reaction). In some embodiments about 0.004 units ofpyrophosphate is added to the reaction mixture.

B. Targeted WGA

In certain embodiments, targeted whole genome amplification (TWGA) isemployed as part of the present invention. Targeted WGA is described,for example, in U.S. Pat. Pub. 20100035232, herein incorporated byreference.

Target Genomes for Design of Targeted Whole Genome Amplification Primers

In some preferred embodiments, one or more target genomes are chosen.The choice of target genomes is dictated by the objective of theanalysis. For example, if the desired outcome of the targeted wholegenome amplification process is to obtain nucleic acid representing thegenome of a biowarfare organism such as Bacillus anthracis, which issuspected of being present in a soil sample at the scene of a biowarfareattack, one may choose to select the genome of Bacillus anthracis as theone and only target genome. If, on the other hand, the desired outcomeof the targeted whole genome amplification process is to obtain nucleicacid representing a group of bacteria, such as, a group of potentialbiowarfare agents, more than one target genome may be selected such as,a group comprising any or all of the following bacteria: Bacillusanthracis, Francisella tularensis, Yersinia pestis, Brucella sp.,Burkholderia mallei, Rickettsia prowazekii, and Escherichia coli 0157.Likewise, a different genome or group of genomes could be selected asthe target genome(s) for other purposes. For example, a human genome ormitochondrial DNA may be the target over common genomes found in a soilsample or other sample environments where a crime may have taken place.Thus, the current methods and compositions can be applied and the humangenome (target) selectively amplified over the background genomes. Otherexamples could include the genomes of group of viruses that causerespiratory illness, pathogens that cause sepsis, or a group of fungiknown to contaminate households.

Background Genomes for Design of Targeted Whole Genome AmplificationPrimers

Background genomes may be selected based on the likelihood of thenucleic acid of certain organisms being present. For example, a soilsample which was handled by a human would be expected to contain nucleicacid representing the genomes of organisms including, but not limitedto: Homo sapiens, Gallus gallus, Guillardia theta, Oryza sativa,Arabidopsis thaliana, Yarrowia lipolytica, Saccharomyces cerevisiae,Debaryomyces hansenii, Kluyveromyces lactis, Schizosaccharmyces pom,Aspergillus fumigatus, Cryptococcus neoformans, Encephalitozooncuniculi, Eremothecium gossypii, Candida glabrata, Apis mellifera,Drosophila melanogaster, Tribolium castaneum, Anopheles gambiae, andCaenorhabditis elegans. Any or all of these genomes are appropriate toestimate as background genomes in the sample. The organisms actually inany particular sample will vary for each sample based upon the sourceand/or environment. Therefore, background genomes may be selected basedupon the identities of organisms actually present in the sample. Thecomposition of a sample can be determined using any of a number oftechniques known to those ordinarily skilled in the art. In a furtherembodiment, the primers can be designed based upon actual identificationof one or more background organisms in the sample, and based uponlikelihood of any further one or more background organisms being in thesample.

Identification of Unique Genome Sequence Segments as PrimerHybridization Sites

Once the target and background genomes of a sample are determined, thenext step is to identify genome sequence segments within the targetgenome which are useful as primer hybridization sites. The efficiency ofa given targeted whole genome amplification is dependent on effectiveuse of primers. To produce an amplification product representative of awhole genome, the primer hybridization sites should have appropriateseparation across the length of the genome. Preferably the meanseparation distance between the primer hybridization sites is about 1000nucleobases or less. More preferably the mean separation is about 800nucleobases in length or less. Even more preferably, the mean separationis about 600 nucleobases in length or less. Most preferably, the meanseparation between primer hybridization sites is about 500 nucleobasesin length or less.

One with ordinary skill in the art will recognize that effective primingfor whole genome amplification depends upon several factors such as thefidelity and processivity of the polymerase enzyme used for primerextension. A longer mean separation distance between primerhybridization sites becomes more acceptable if the polymerase enzyme hashigh processivity. This indicates that the polymerase binds tightly tothe nucleic acid template. This is a desirable characteristic fortargeted whole genome amplification because it enables the polymerase toremain bound to the template nucleic acid and continue to extend thecomplementary nucleic acid strand being synthesized. Examples ofpolymerase enzymes having high processivity include, but are not limitedto Phi29 polymerase and Taq polymerase. Protein engineering strategieshave been used to produce high processivity polymerase enzymes, forexample, by covalent linkage of a polymerase to a DNA-binding protein(Wang et al., Nucl. Acids Res., 2004, 32(3) 1197-1207, hereinincorporated by reference). As polymerases with improved processivitybecome available, longer mean separation distances, even greatlyexceeding 1000 nucleobases may be acceptable for targeted whole genomeamplification.

Hybridization Sensitivity and Selectivity

For the purpose of targeted whole genome amplification, the choice oflength of the primer hybridization sites (genome sequence segments) andthe lengths of the corresponding primers hybridizing thereto, preferablywill balance two factors; (1) sensitivity, which indicates the frequencyof binding of a given primer to the target genome, and (2) selectivity,which indicates the extent to which a given primer hybridizes to thetarget genome with greater frequency than it hybridizes to backgroundgenomes. Generally, longer primers tend toward greater selectivity andlesser sensitivity while the converse holds for shorter primers.Preferably primers of about 5 to about 13 nucleobases in length areuseful for targeted whole genome amplification; however, primer lengthsfalling outside of this range can be used as well. One will recognizethat this range comprises primers having lengths of 5, 6, 7, 8, 9, 10,11, 12 and 13 nucleobases. Primer size affects the balance betweenselectivity of the primer and sensitivity of the primer. Optimal primerlength is determined for each sample with this balance in mind. Primerswith lengths less than 5 nucleobases or greater than 13 nucleobases arealso useful if the selectivity and sensitivity can be optimallymaintained for that sample. Choosing a plurality of primers havingvarious lengths provide broad priming across the target genomesequence(s) while also providing preferential binding of the primers tothe target genome sequence(s) relative to the background genomesequences.

Selection Threshold Criteria

In some embodiments, it is preferable to determine a suitable sub-set ofthe total unique genome sequence segments in order to reduce the totalnumber of primers in the targeted whole genome amplification set inorder to reduce the costs and complexity of the primer set. In someembodiments, determination of the suitable sub-set of unique genomesequence segments entails choosing one or more threshold criteria whichindicate a useful and practical cut-off point for sensitivity and/orselectivity of a given genome sequence segment. Examples of suchcriteria include, but are not limited to, a selected threshold frequencyof occurrence (a frequency of occurrence threshold value), and aselected selectivity ratio (a selectivity ratio threshold value).

In some embodiments, it is useful to rank the total unique genomesequence segments according to the criteria. For example, the totalunique genome sequence segments are ranked according to frequency ofoccurrence with the #1 rank indicating the greatest frequency ofoccurrence and the lowest rank indicating the lowest frequency ofoccurrence. A threshold frequency of occurrence can then be chosen fromthe ranks. The threshold frequency of occurrence serves as the dividingline between members of the sub-set chosen for further analysis and themembers that will not be further analyzed.

Design of Primers

The primers that are designed to hybridize to the selected genomesequence segments are preferably 100% complementary to the genomesequence segments. In other embodiments, the primers that are designedto hybridize to the selected genome sequence segments are at least about70% to about 100% complementary to the genome sequence segments, or anywhole or fractional number therebetween. In general terms, design ofprimers for hybridization to selected nucleic acid sequences is wellknown to those with skill in the art and can be aided by commerciallyavailable computer programs. It is generally preferable to design agiven primer such that it is the same length as the genome sequencesegment which was analyzed and chosen as a primer hybridization site.However, in some cases it may be advantageous to alter the length of theprimer relative to the primer hybridization site. For example, if theprimer is analyzed and found to have an unfavorable melting temperatureand would benefit from elongation at the 5′ or 3′ end to produce aprimer having an improved affinity for the target genome sequence. Thelength of the primer can be either increased or decreased. One withordinary skill will recognize that alteration of the primer length alsoalters the primer hybridization site so that it no longer identical tothe originally selected genome sequence segment. In some cases, it maybe beneficial to analyze the genome sequence segment which correspondsto the hybridization site of a given length-altered primer. Thisanalysis may be done by examination of data including but not limitedto: frequency of occurrence and selectivity ratio and may also be doneby actual in vitro testing of the length-altered primer.

In some embodiments, in cases where it may be advantageous to design aprimer to be less than 100% complementary to its corresponding genomesequence segment, it is also advantageous to examine the complement ofthe re-calculate selection criteria (such as frequency of occurrence andselectivity ratio) for a hypothetical genome sequence segment that is100% complementary to the primer which is less than 100% complementaryto its corresponding original genome sequence segment. If the selectioncriteria are unfavorable, it would be advantageous to consider design ofan alternate primer sequence having improved selection criteria.

III. Sequencing Technologies

As described above, embodiments of the present invention involvesequencing the DNA library that is generated with the microfluidicscards. The present invention is not limited by the type of sequencingmethod employed. Exemplary sequencing methods are described below.

Illustrative non-limiting examples of nucleic acid sequencing techniquesinclude, but are not limited to, chain terminator (Sanger) sequencingand dye terminator sequencing. Chain terminator sequencing usessequence-specific termination of a DNA synthesis reaction using modifiednucleotide substrates. Extension is initiated at a specific site on thetemplate DNA by using a short radioactive, or other labeled,oligonucleotide primer complementary to the template at that region. Theoligonucleotide primer is extended using a DNA polymerase, standard fourdeoxynucleotide bases, and a low concentration of one chain terminatingnucleotide, most commonly a di-deoxynucleotide. This reaction isrepeated in four separate tubes with each of the bases taking turns asthe di-deoxynucleotide. Limited incorporation of the chain terminatingnucleotide by the DNA polymerase results in a series of related DNAfragments that are terminated only at positions where that particulardi-deoxynucleotide is used. For each reaction tube, the fragments aresize-separated by electrophoresis in a slab polyacrylamide gel or acapillary tube filled with a viscous polymer. The sequence is determinedby reading which lane produces a visualized mark from the labeled primeras you scan from the top of the gel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Completesequencing can be performed in a single reaction by labeling each of thedi-deoxynucleotide chain-terminators with a separate fluorescent dye,which fluoresces at a different wavelength.

A set of methods referred to as “next-generation sequencing” techniqueshave emerged as alternatives to Sanger and dye-terminator sequencingmethods (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLeanet al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated byreference in their entirety). Most current methods describe the use ofnext-generation sequencing technology for de novo sequencing of wholegenomes to determine the primary nucleic acid sequence of an organism.In addition, targeted re-sequencing (deep sequencing) allows forsensitive mutation detection within a population of wild-type sequence.Some examples include recent work describing the identification of HIVdrug-resistant variants as well as EGFR mutations for determiningresponse to anti-TK therapeutic drugs. Recent publications describingthe use of bar code primer sequences permit the simultaneous sequencingof multiple samples during a typical sequencing run including, forexample: Margulies, M. et al. “Genome Sequencing in MicrofabricatedHigh-Density Picoliter Reactors”, Nature, 437, 376-80 (2005); Mikkelsen,T. et al. “Genome-Wide Maps of Chromatin State in Pluripotent andLineage-Committed Cells”, Nature, 448, 553-60 (2007); McLaughlin, S. etal. “Whole-Genome Resequencing with Short Reads: Accurate MutationDiscovery with Mate Pairs and Quality Values”, ASHG Annual Meeting(2007); Shendure J. et al. “Accurate Multiplex Polony Sequencing of anEvolved Bacterial Genome”, Science, 309, 1728-32 (2005); Harris, T. etal. “Single-Molecule DNA Sequencing of a Viral Genome”, Science, 320,106-9 (2008); Simen, B. et al. “Prevalence of Low Abundance DrugResistant Variants by Ultra Deep Sequencing in Chronically HIV-infectedAntiretroviral (ARV) Naïve Patients and the Impact on VirologicOutcomes”, 16th International HIV Drug Resistance Workshop, Barbados(2007); Thomas, R. et al. “Sensitive Mutation Detection in HeterogeneousCancer Specimens by Massively Parallel Picoliter Reactor Sequencing”,Nature Med., 12, 852-855 (2006); Mitsuya, Y. et al. “Minority HumanImmunodeficiency Virus Type 1 Variants in Antiretroviral-Naïve Personswith Reverse Transcriptase Codon 215 Revertant Mutations”, J. Vir., 82,10747-10755 (2008); Binladen, J. et al. “The Use of Coded PCR PrimersEnables High-Throughput Sequencing of Multiple Homolog AmplificationProducts by 454 Parallel Sequencing”, PLoS ONE, 2, e197 (2007); andHoffmann, C. et al. “DNA Bar Coding and Pyrosequencing to Identify RareHIV Drug Resistance Mutations”, Nuc. Acids Res., 35, e91 (2007), all ofwhich are herein incorporated by reference.

Compared to traditional Sanger sequencing, next-gen sequencingtechnology produces large amounts of sequencing data points. A typicalrun can easily generate tens to hundreds of megabases per run, with apotential daily output reaching into the gigabase range. This translatesto several orders of magnitude greater than a standard 96-well plate,which can generate several hundred data points in a typical multiplexrun. Target amplicons that differ by as little as one nucleotide caneasily be distinguished, even when multiple targets from related speciesare present. This greatly enhances the ability to do accurategenotyping. Next-gen sequence alignment software programs used toproduce consensus sequences can easily identify novel point mutations,which could result in new strains with associated drug resistance. Theuse of primer bar coding also allows multiplexing of different patientsamples within a single sequencing run.

Next-generation sequencing (NGS) methods share the common feature ofmassively parallel, high-throughput strategies, with the goal of lowercosts in comparison to older sequencing methods. NGS methods can bebroadly divided into those that require template amplification and thosethat do not. Amplification-requiring methods include pyrosequencingcommercialized by Roche as the 454 technology platforms (e.g., GS 20 andGS FLX), the Solexa platform commercialized by Illumina, and theSupported Oligonucleotide Ligation and Detection (SOLiD) platformcommercialized by Applied Biosystems. Non-amplification approaches, alsoknown as single-molecule sequencing, are exemplified by the HeliScopeplatform commercialized by Helicos BioSciences, and emerging platformscommercialized by VisiGen and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated byreference in its entirety), template DNA is fragmented, end-repaired,ligated to adaptors, and clonally amplified in-situ by capturing singletemplate molecules with beads bearing oligonucleotides complementary tothe adaptors. Each bead bearing a single template type iscompartmentalized into a water-in-oil microvesicle, and the template isclonally amplified using a technique referred to as emulsion PCR. Theemulsion is disrupted after amplification and beads are deposited intoindividual wells of a picotiter plate functioning as a flow cell duringthe sequencing reactions. Ordered, iterative introduction of each of thefour dNTP reagents occurs in the flow cell in the presence of sequencingenzymes and luminescent reporter such as luciferase. In the event thatan appropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 1×10⁶ sequencereads can be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S.Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488;each herein incorporated by reference in its entirety), sequencing dataare produced in the form of shorter-length reads. In this method,single-stranded fragmented DNA is end-repaired to generate5′-phosphorylated blunt ends, followed by Klenow-mediated addition of asingle A base to the 3′ end of the fragments. A-addition facilitatesaddition of T-overhang adaptor oligonucleotides, which are subsequentlyused to capture the template-adaptor molecules on the surface of a flowcell that is studded with oligonucleotide anchors. The anchor is used asa PCR primer, but because of the length of the template and itsproximity to other nearby anchor oligonucleotides, extension by PCRresults in the “arching over” of the molecule to hybridize with anadjacent anchor oligonucleotide to form a bridge structure on thesurface of the flow cell. These loops of DNA are denatured and cleaved.Forward strands are then sequenced with reversible dye terminators. Thesequence of incorporated nucleotides is determined by detection ofpost-incorporation fluorescence, with each fluor and block removed priorto the next cycle of dNTP addition. Sequence read length ranges from 36nucleotides to over 50 nucleotides, with overall output exceeding 1billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No.6,130,073; each herein incorporated by reference in their entirety) alsoinvolves fragmentation of the template, ligation to oligonucleotideadaptors, attachment to beads, and clonal amplification by emulsion PCR.Following this, beads bearing template are immobilized on a derivatizedsurface of a glass flow-cell, and a primer complementary to the adaptoroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color and thus identity of each probe corresponds tospecified color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In certain embodiments, nanopore sequencing in employed (see, e.g.,Astier et al., Am Chem. Soc. 2006 Feb. 8; 128(5):1705-10, hereinincorporated by reference). The theory behind nanopore sequencing has todo with what occurs when the nanopore is immersed in a conducting fluidand a potential (voltage) is applied across it: under these conditions aslight electric current due to conduction of ions through the nanoporecan be observed, and the amount of current is exceedingly sensitive tothe size of the nanopore. If DNA molecules pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore, thereby allowing thesequences of the DNA molecule to be determined. The nanopore may be asolid-state pore fabricated on a metal and/or nonmetal surface, or aprotein-based nanopore, such as α-hemolysin (Clarke et al., Nat.Nanotech., 4, Feb. 22, 2009: 265-270).

HeliScope by Helicos BioSciences (Voelkerding et al., Clinical Chem.,55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296;U.S. Pat. No. 7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No.7,482,120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat.No. 6,911,345; U.S. Pat. No. 7,501,245; each herein incorporated byreference in their entirety) is the first commercialized single-moleculesequencing platform. This method does not require clonal amplification.Template DNA is fragmented and polyadenylated at the 3′ end, with thefinal adenosine bearing a fluorescent label. Denatured polyadenylatedtemplate fragments are ligated to poly(dT) oligonucleotides on thesurface of a flow cell. Initial physical locations of captured templatemolecules are recorded by a CCD camera, and then label is cleaved andwashed away. Sequencing is achieved by addition of polymerase and serialaddition of fluorescently-labeled dNTP reagents. Incorporation eventsresult in fluor signal corresponding to the dNTP, and signal is capturedby a CCD camera before each round of dNTP addition. Sequence read lengthranges from 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run. Other emerging single moleculesequencing methods real-time sequencing by synthesis using a VisiGenplatform (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; U.S.Pat. No. 7,329,492; U.S. patent application Ser. No. 11/671,956; U.S.patent application Ser. No. 11/781,166; each herein incorporated byreference in their entirety) in which immobilized, primed DNA templateis subjected to strand extension using a fluorescently-modifiedpolymerase and florescent acceptor molecules, resulting in detectiblefluorescence resonance energy transfer (FRET) upon nucleotide addition.Another real-time single molecule sequencing system developed by PacificBiosciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.7,170,050; U.S. Pat. No. 7,302,146; U.S. Pat. No. 7,313,308; U.S. Pat.No. 7,476,503; all of which are herein incorporated by reference)utilizes reaction wells 50-100 nm in diameter and encompassing areaction volume of approximately 20 zeptoliters (10×10⁻²¹ L). Sequencingreactions are performed using immobilized template, modified phi29 DNApolymerase, and high local concentrations of fluorescently labeleddNTPs. High local concentrations and continuous reaction conditionsallow incorporation events to be captured in real time by fluor signaldetection using laser excitation, an optical waveguide, and a CCDcamera.

In certain embodiments, the single molecule real time (SMRT) DNAsequencing methods using zero-mode waveguides (ZMWs) developed byPacific Biosciences, or similar methods, are employed. With thistechnology, DNA sequencing is performed on SMRT chips, each containingthousands of zero-mode waveguides (ZMWs). A ZMW is a hole, tens ofnanometers in diameter, fabricated in a 100 nm metal film deposited on asilicon dioxide substrate. Each ZMW becomes a nanophotonic visualizationchamber providing a detection volume of just 20 zeptoliters (10-21liters). At this volume, the activity of a single molecule can bedetected amongst a background of thousands of labeled nucleotides.

The ZMW provides a window for watching DNA polymerase as it performssequencing by synthesis. Within each chamber, a single DNA polymerasemolecule is attached to the bottom surface such that it permanentlyresides within the detection volume. Phospholinked nucleotides, eachtype labeled with a different colored fluorophore, are then introducedinto the reaction solution at high concentrations which promote enzymespeed, accuracy, and processivity. Due to the small size of the ZMW,even at these high, biologically relevant concentrations, the detectionvolume is occupied by nucleotides only a small fraction of the time. Inaddition, visits to the detection volume are fast, lasting only a fewmicroseconds, due to the very small distance that diffusion has to carrythe nucleotides. The result is a very low background.

As the DNA polymerase incorporates complementary nucleotides, each baseis held within the detection volume for tens of milliseconds, which isorders of magnitude longer than the amount of time it takes a nucleotideto diffuse in and out of the detection volume. During this time, theengaged fluorophore emits fluorescent light whose color corresponds tothe base identity. Then, as part of the natural incorporation cycle, thepolymerase cleaves the bond holding the fluorophore in place and the dyediffuses out of the detection volume. Following incorporation, thesignal immediately returns to baseline and the process repeats.

Unhampered and uninterrupted, the DNA polymerase continues incorporatingbases at a speed of tens per second. In this way, a completely naturallong chain of DNA is produced in minutes. Simultaneous and continuousdetection occurs across all of the thousands of ZMWs on the SMRT chip inreal time. Researchers at PacBio have demonstrated this approach has thecapability to produce reads thousands of nucleotides in length.

EXAMPLES

The following Examples are presented in order to provide certainexemplary embodiments of the present invention and are not intended tolimit the scope thereof.

Example 1 Stabilized Enzyme Mixtures

This example describes the development of stabilized enzyme mixturesthat are useful in methods such as whole genome amplification (WGA). Asdescribed in FIG. 15, many excipient formulations were tested forcompatibility with the WGA of Phi-29, Polymerase I, and inorganicpyrophosphatase. It was found that 16 proprietary stabilizationformulations (called excipients) were not useful at successfullystabilizing the enzymes generally used in WGA. It was found that BSA wasan important component for stabilizing these enzymes, as shown in FIG.16.

One preferred excipient that was developed is called “Ibis Formula 33.”A description of how to make this formulation is as follows. A mixtureof 8968 u/ml Phi-29 polymerase (Monserate), 180 u/ml Polymerase I(Epicentre Biotechnologies), 3.6 u/ml Inorganic Pyrophosphatase (U.S.Biochemical) is mixed 1:1 with a solution containing 20% trehalose, 10mM (NH₄)₂SO₄, 12 MgCl₂, 50 mM Tris pH 7.6, 0.05% Tween 40, 4 mM DTT andBSA at a level to achieve a final concentration of 0.25% (due to volumechanges during dialysis there is a higher concentration of BSA duringdialysis so that the solution can be brought up to the correct finalvolume post-dialysis.) This mixture is dialyzed using a 30 KDa dialysismembrane into a solution containing 20% trehalose, 10 mM (NH₄)₂SO₄, 12MgCl₂, 50 mM Tris pH 7.6, 0.05% Tween 40 and 4 mM DTT for 4 hours atroom temperature.

The dialyzed enzyme mixture is then removed from the dialysis membraneand brought up to the correct final volume (the level of dilution duringthe dialysis/dilution process controls how much enzyme is present perlyophilized unit) using a solution containing 20% trehalose, 10 mM(NH₄)₂SO₄, 12 MgCl₂, 50 mM Tris pH 7.6, 0.05% Tween 40 and 4 mM DTT.This solution is then aliquoted into appropriate volumes, frozen andsubjected to a high vacuum to remove the water via sublimation togenerate a lyophilized composition.

A number of BSA levels (0.13%-1% final concentration) as well as theaddition of 0.5% (final concentration) of PEG-8000 were tested using thesame general scheme described above. The results of this testing areshown in FIG. 17.

Using the excipients developed in this work we successfully demonstratedthat the enzymatic activity of all three of these enzymes is maintainedduring lyophilization and the stability of these enzymes wasdramatically increased from less than 10 days at room temperature (−25C) to more than 2 months at 40 C. This formulation also increases thetotal reaction yield when compared to a fresh (i.e., stored in a −20 Cfreezer until use) enzyme mix. Long-term stability results for storageat room temperature are shown in FIG. 18. This figures shows that thelyophilized enzymes in Ibis Formula 33 when stored at room temperaturefor 4 months performed equal to fresh enzyme. FIG. 19 shows long-termstorage stability using accelerated aging of 40 degrees Celsius for twomonths, which is equal to approximately 6 months of room temperaturestorage. FIG. 19 shows that the lyophilized enzymes in Ibis Formula 33performed equal to fresh enzyme after two months at 40 degrees Celsius.

Example 2 Sample Preparation Methods

This example describes various sample preparation methods that may beused, for example, in microfluidic devices. Such methods allows the useof a single universal buffer and make it possible for the sample toremain in single tube. The general outline for such methods include thefollowing steps: lysis and extraction; whole-genome amplification (orother amplification method); fragmentation of DNA; end polishing;ligation; removal of incomplete products; and final clean-up. Describedbelow are certain details on a number of such steps.

i. Exemplary Fragmentation Methods

An amplified sample (e.g., WGA sample) can be fragmented with a devicecomposed of the following components: 1) sonicator assembly: 2.4meghaertz miniature ultrasonic nebulizer, Sonaer 241V; 2) Transducer:gold coated nebulizer crystal, Sonaer 24AU; 3) Power supply: 24 voltpower supply, Sonaer ST624; and 4) Lid: machined plastic to createenclosure in sonicator assembly. Sonication using this method can beconducted for 10 minutes total time, with 50% duty cycle with intervalsof 15 s on, 15 s off.

ii. Ligation Reaction Speed Optimized

The speed of the ligation was optimized from an initial condition, whichallowed a significant increase in the amount of final product generatedin 30 minutes. The optimization included modified buffer components orconcentrations and modified enzyme concentrations. The parameters areshown in Table 1 below. The reactions conditions were 30 C, 30 minutes,and 65 C, 10 minutes.

TABLE 1 Tween T4 Ligase trehalose (NH4)2SO4 Tris MgC12 40 DTT condition1  3,000 units 452 5 mM 8 mM 40 mM 9.6 mM 1% 2.81 mM optimized 10,000units 452 5 mM 8 mM 40 mM 9.6 mM 1% 2.81 mM dNTPs WGA (each) ATP PEG8000 pH Hairpin Insert Primers condition 1 518 uM 3 mM 0 7.6 4.36 uM0.436 uM 35.8 uM optimized 518 uM 334 uM   4.55% 7.6 4.36 uM 0.436 uM35.8 uMImportant parameters for optimization included: ligase concentration,ATP concentration, PEG concentration, addition of sodium pyrophosphateafter 1 minute of reaction. The optimized conditions lead to asignificant increase in final product after 30 minutes.

iii. WGA Enzymes in Amplification Buffer End Polish Efficiently

Conditions were examined to test end polishing efficiency. Conditionstested included: reaction time, reaction temperature, dATPconcentration, DTT concentration, PEG concentration, sperimidineconcentration, poly-lysine concentration. The parameters employed areshown in Table 2 below. The reaction conditions employed were: 37 C, 5minutes; 50 C, 2 minutes; and 75 C, 10 minutes.

TABLE 2 Phi 29 Klenow exo- trehalose (NH4)2SO4 Tris MgCl2 Tween 40 end100 units 40 units 452 5 mM 8 mM 40 mM 9.6 mM 1% polish dNTPs WGA DuplexDTT (each) dATP pH Primers Oligo end 2.81 mM 518 uM 5 mM 7.6 35.8 uM 1uM polishThe analysis method employed was electro-spray ionization time of flightmass spectrometry (ESI-TOF MS). One important parameter fond wasreaction temperature (this was an important parameter as below 55 Cminimal A-tailing was observed while above 55 C significant A-tailingwas observed), dATP concentration (increasing the dATP concentrationincreases A-tailing). The results found that 100% of the products wereblunted appropriately, and approximately 70% of the ends were A-tailed.

iv. Exonuclease Clean-Up of Ligation Reactions

A ligation reaction performed using ‘condition 1’ shown in Table 1 above(with a slightly different hairpin/insert sequences) digested using exoIII and exo VII. The hairpin and insert concentration shown below areprior to the ligation reaction. The majority of the insert was convertedto “final product” during the ligation reaction. The conditions used areshown in Table 3. The reactions conditions were 37 C, 1 hr, and 70 C, 10minutes.

TABLE 3 Exo III Exo VII trehalose (NH4)2SO4 Tris MgCl2 Tween 40 exo 100units 5 units 452 5 mM 8 mM 40 mM 9.6 mM 1% digest dNTPs WGA DTT (each)pH Hairpin Insert Primers exo 2.81 mM 518 uM 7.6 4.36 uM 0.436 uM 35.8uM digest

It is noted that WGA amplification primers were included in all thesereactions even though they are not part of the reaction to determine ifthey would cause any inhibition (none was observed.) Similarly, dNTPswere included in reactions (such as the ligation or exo digestion) eventhough they were not needed in the reaction to determine if they wouldcause any inhibition (none was observed.) The exonuclease degradesnon-circularized templates, removing any DNA other than final product.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A microfluidic card, comprising: a) a loading port configured for introduction of a biological sample; b) a nucleic acid extraction sub-circuit wherein said nucleic acid extraction sub-circuit comprises: i) a nucleic acid extraction component configured to bind nucleic acids present in said biological sample; ii) a wash buffer; iii) an elution buffer; and iv) a component configured for moving said elution buffer over said nucleic acid extraction component to generate a mixture of extracted nucleic acids; and c) an amplification sub-circuit, wherein said amplification sub-circuit comprises a stabilized enzyme mixture, wherein said enzyme mixture comprises at least one amplification-related enzyme useful for performing amplification on said extracted nucleic acid to generate amplified nucleic acid, d) a fragmentation sub-circuit, wherein said fragmentation sub-circuit comprises: i) a reagent mixture configured for digesting nucleic acid to generate fragmented nucleic acid; and/or ii) a fragmentation component configured for mechanically fragmenting nucleic acid to generate fragmented nucleic acid; and iii) at least one type of enzyme configured for polishing the ends of said fragmented nucleic acid; and e) a linker sub-circuit, wherein said linker ligation sub-circuit comprises: i) nucleic acid linkers configured for use in sequencing methods; ii) a ligation enzyme mixture configured for ligating said nucleic acid linkers to nucleic acid to generate a nucleic acid sequencing library; and iii) at least one type of enzyme configured for polishing the ends of said nucleic acid sequencing library; and f) a purification sub-circuit, wherein said purification sub-circuit comprises a nucleic acid purification component.
 2. The microfluidic card of claim 1, further comprising: g) a lysis sub-circuit operably linked to said loading port, wherein said lysis sub-circuit comprises: i) a lysis chamber; and ii) a lysis buffer wherein said lysis sub-circuit is configured to lyse said biological sample with said lysis buffer to generate a lysed sample.
 3. The microfluidic card of claim 1, wherein said at least one amplification-related enzyme comprises an enzyme useful for performing whole genome amplification (WGA), PCR, or TMA.
 4. The microfluidic card of claim 1, wherein said at least one amplification-related enzyme is selected from the group consisting of: Phi-29 polymerase, E. coli DNA polymerase I, inorganic pyrophosphatase, or any combination thereof.
 5. The microfluidic card of claim 1, wherein said stabilized enzyme mixture further comprises: i) BSA, ii) a sugar, and iii) at least one additional component selected from the group consisting of: an inorganic salt, a divalent metal cation, a buffering agent, an emulsifier, and a reducing agent.
 6. The microfluidic card of claim 1, wherein said stabilized enzyme mixture further comprises: i) BSA, ii) a sugar, iii) an inorganic salt, iv) a divalent metal cation, v) a buffering agent, vi) an emulsifier, vii) and a reducing agent.
 7. The microfluidic card of claim 1, further comprising an outlet port configured to allow a user to withdraw at least a portion of said nucleic acid sequencing library.
 8. The microfluidic card of claim 5, wherein said BSA is present at a concentration of 0.05%-3.0%, said sugar is present at a concentration of 5-35%, said inorganic salt is present at a concentration of 1 mM-25 mM, said divalent metal cation is present at a concentration of 1 mM-30 mM, said buffering agent is present at a concentration of 10 mM-100 mM, said emulsifier is present at a concentration of 0.01%-0.15%, and said reducing agent is present at a concentration of 1 mM-10 mM in said stabilized enzyme mixture.
 9. The microfluidic card of claim 2, further comprising a waste chamber operably linked to one or more of said lysis sub-circuit, said nucleic acid extraction sub-circuit, said amplification sub-circuit, said fragmentation sub-circuit, and/or said linker ligation sub-circuit and said purification sub-circuit.
 10. The microfluidic card of claim 1, further comprising a processing instrument configured for passing buffers and samples.
 11. The microfluidic card of claim 1, further comprising one or more sealed packages comprising one or more of said lysis buffer, said wash buffer, said elution buffer, said stabilized enzyme mixture, said reagent mixture configured for digesting said amplified nucleic acid, and said ligation enzyme mixture.
 12. The microfluidic card of claim 2, wherein in said lysis sub-circuit, said nucleic acid extraction sub-circuit, said amplification sub-circuit, said fragmentation sub-circuit, said linker ligation sub-circuit, and said purification sub-circuit are operably linked in sequence.
 13. The microfluidic card of claim 2, wherein said lysis buffer, said wash buffer, and said elution buffer are a single buffer.
 14. A system comprising an automated nucleic acid sequencer and a microfluidic card of claim 7, wherein said outlet port of said microfluidic card is in operable fluid communication with said nucleic acid sequencer.
 15. The microfluidic card of claim 1, wherein said linker is a nucleic acid adaptor. 